Exploring TTN variants as genetic insights into cardiomyopathy pathogenesis and potential emerging clues to molecular mechanisms in cardiomyopathies

The giant protein titin (TTN) is a sarcomeric protein that forms the myofibrillar backbone for the components of the contractile machinery which plays a crucial role in muscle disorders and cardiomyopathies. Diagnosing TTN pathogenic variants has important implications for patient management and genetic counseling. Genetic testing for TTN variants can help identify individuals at risk for developing cardiomyopathies, allowing for early intervention and personalized treatment strategies. Furthermore, identifying TTN variants can inform prognosis and guide therapeutic decisions. Deciphering the intricate genotype–phenotype correlations between TTN variants and their pathologic traits in cardiomyopathies is imperative for gene-based diagnosis, risk assessment, and personalized clinical management. With the increasing use of next-generation sequencing (NGS), a high number of variants in the TTN gene have been detected in patients with cardiomyopathies. However, not all TTN variants detected in cardiomyopathy cohorts can be assumed to be disease-causing. The interpretation of TTN variants remains challenging due to high background population variation. This narrative review aimed to comprehensively summarize current evidence on TTN variants identified in published cardiomyopathy studies and determine which specific variants are likely pathogenic contributors to cardiomyopathy development.


Variant annotation and pathogenicity assessment
The annotation of TTN variants involved a comprehensive pathogenicity assessment using multiple tools.This included the application of the American College of Medical Genetics and Genomics (ACMG) guidelines, consultation of ClinVar for variant interpretation, insights from Mutation Taster regarding potential pathogenicity, the use of the Combined Annotation Dependent Depletion (CADD) scoring system for deleteriousness prediction, and evaluation through Genomic Evolutionary Rate Profiling (GERP) to assess evolutionary conservation which are explain more in the following.
We determineded the ACMG score for each variant using franklin, an online database (https:// frank lin.genoox.com/ clini cal-db).After adding the name in this website, varints ACMG score anongside with other features are provided.

ACMG score
The American College of Medical Genetics and Genomics (ACMG) previously established guidelines for interpreting sequence variants.With the rapid advancements in sequencing technology over the past decade, this report suggests the adoption of standardized terms such as "pathogenic, " "likely pathogenic, " "uncertain significance, " "likely benign, " and "benign" to characterize variants found in genes associated with Mendelian disorders.Additionally, the recommendation outlines a systematic approach for classifying variants into these categories, relying on various types of evidence, including population data (Population, disease-specific, and sequence databases), computational data (using silico tools for missense prediction, splice site prediction and nucleotide conservation prediction), functional data, and segregation data 15,16 .
In this classification a variant is considered pathogenic if it meets the requirement of having a very strong criterion (PVS1) along with at least one strong criterion (PS1-PS4), or alternatively, two or more moderate criteria (PM1-PM6), or a combination of one moderate criterion and one supporting criterion (PP1-PP5).Another condition is that a variant can be classified as pathogenic if it satisfies the condition of having at least two strong criteria (PS1-PS4).Additionally, a variant can be considered pathogenic if it meets the criteria of having one strong criterion (PS1-PS4) and either three moderate criteria (PM1-PM6), two moderate criteria and at least two supporting criteria (PP1-PP5), or one moderate criterion and at least four supporting criteria (PP1-PP5) 16 .
A variant is considered likely pathogenic if it satisfies the condition of having one very strong criterion (PVS1) in combination with one moderate criterion (PM1-PM6).Alternatively, a likely pathogenic variant may exhibit one strong criterion (PS1-PS4) along with one to two moderate criteria (PM1-PM6).Another criterion designates a variant as likely pathogenic if it possesses one strong criterion (PS1-PS4) and at least two supporting criteria (PP1-PP5).Furthermore, likely pathogenic variants may be identified if they meet the requirement of having three or more moderate criteria (PM1-PM6).Additionally, a variant is classified as likely pathogenic if it has two moderate criteria (PM1-PM6) and at least two supporting criteria (PP1-PP5), or if it exhibits one moderate criterion (PM1-PM6) along with at least four supporting criteria (PP1-PP5) 16 .More information is provided in "Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology" 16 .
The ACMG score for each variant is determined using Franklin, an online database available at https:// frank lin.genoox.com/ clini cal-db.Upon entering the variant's name on this website, the ACMG score, along with other relevant features, is provided.
www.nature.com/scientificreports/CADD score CADD, or Combined Annotation Dependent Depletion, serves as a tool for evaluating the deleteriousness of various genetic variants, including single nucleotide changes, multi-nucleotide substitutions, and insertion/deletion variants within the human genome.In contrast to many other annotation tools that often rely on a singular type of information or have limited applicability, CADD offers a versatile metric that objectively combines diverse annotations.The framework integrates multiple annotations into a unified metric by comparing variants that have undergone natural selection with simulated mutations.It incorporates information from more than 60 genomic features to assess single nucleotide variants and short insertions and deletions across the reference assembly.The C-scores generated by CADD demonstrate robust correlations with allelic diversity, pathogenicity of coding and non-coding variants, and experimentally measured regulatory effects.Notably, C-scores of variants associated with complex traits in genome-wide association studies (GWAS) are significantly higher than matched controls, showing correlation with study sample size, indicative of improved accuracy in larger GWAS.CADD employs a machine learning model that distinguishes between simulated de novo variants, potentially encompassing neutral or harmful alleles, and variants persisting in human populations since the split from chimpanzees.
CADD's capability to quantitatively prioritize functional, deleterious, and disease-causing variants spans a wide range of functional categories, effect sizes, and genetic architectures.This tool enhances the scoring of coding variants through features derived from the ESM-1v protein language model and improves the scoring of regulatory variants using features from a convolutional neural network trained on open chromatin regions.For more information CADD has been detailed in four publications [17][18][19][20] .

MutationTaster
MutationTaster is a web-based application designed to assess the disease-causing potential of DNA sequence variants.It employs in silico tests to estimate the impact of a variant on the gene product or protein, conducting assessments at both the protein and DNA levels.Unlike tools limited to single amino acid substitutions, Muta-tionTaster can handle a variety of variants, including synonymous and intronic ones 21 .The software, written in Perl programming language and utilizes integrated databases (Ensembl, UniProt, ClinVar, ExAC, 1000 Genomes Project, phyloP and phastCons) to filter out known harmless polymorphisms.Various tests, such as amino acid substitution, conservation, domain functionality, splicing effects, and regulatory element abrogation, are performed on the remaining single-nucleotide polymorphisms (SNPs).The results are evaluated by a Naive Bayes classifier, and the output indicates whether the alteration is known or predicted to be harmless or disease-causing, providing detailed information about the mutation.While the tool demonstrates a raw accuracy of approximately 90%, considering knowledge about common polymorphisms and known disease mutations significantly improves the rate of correct classifications.However, it is important to note that predictions of clinical effects suffer from a lack of specificity, a common constraint across various prediction methods 22,23 .

GERP
Comparative genomic approaches have historically identified mutation sites under purifying selection by examining conserved sequences across distantly related species.Additionally, the performance of such approaches may be limited for short-lived functional elements that don't exhibit sequence conservation across numerous species.Genomic Evolutionary Rate Profiling (GERP) score is associated with the strength of selection (Nes).Results indicate that the GERP score is linked to the intensity of purifying selection.Nevertheless, variations in selection coefficients or turnover of functional elements over time can significantly impact the GERP distribution, leading to unexpected relationships between GERP and Nes 24 .The GERP score is characterized as the decrease in the count of substitutions in the multi-species sequence alignment in comparison to the neutral expectation.GERP ++ scores span from − 12.3 to 6.17, with elevated scores signifying a greater level of evolutionary constraint.

Data integration
Data integration encompassed the consolidation of relevant information, including Position on Chromosome, HGVS DNA, HGVS Protein, exon or intron number, and dbSNP identifiers.Rigorous quality control measures were then applied to ensure the accuracy and consistency of data extraction and annotation.

Statistical analysis
Descriptive statistics were employed for a comprehensive analysis, summarizing the distribution of TTN variants in terms of positions, types, and associated pathogenicity.

Ethical considerations
Ethical considerations are considered in the study, with a commitment to adhering to Data reliability and responsible data handling.In the present study, it is important to note that no human subjects were involved, as this investigation is a comprehensive review rather than an experimental study.The research focused on analyzing reported variants available on PubMed, and ethical approval or consent from human participants was not applicable.

The molecular structure of titin
The TTN gene located on the second human chromosome in the 2q31 area.This gene contains 364 exons, which their translation produces a 4200-kDa protein with ~ 38,000 amino acid residues, the largest polypeptide found in the human body.The Titin giant protein, also known as connectin, is the third most abundant protein found www.nature.com/scientificreports/ in striated muscle among the vertebrates, after myosin and actin.The Titin is a flexible filament that is more than 1 µm long and 3-4 nm wide and spans half of the sarcomere as the repeating contractile unit that gives striated muscle characteristic striped appearance 25 .
Titin has a complex multidomain structure which is composed of four main structural and functional regions: the N-terminal Z-line acts as an anchor for the sarcomeric Z-disk; the I-band provides elastic properties; the A-band stabilizes the thick filament; and the C-terminal M-line extremity overlaps in an antiparallel orientation with another titin molecule's C-terminus, allowing for modulation of titin expression and turnover via the tyrosine kinase domain 26 .
The N-terminus contains immunoglobulin (Ig) domains, fibronectin (FN) domains, and a Z-disk region 27 .The rest of the titin molecule includes an elastic I-band region, a spring-like Pro-Glu-Val-Lys (PEVK) domain, three unique sequences called Novex 1, 2, and 3, cardiac-specific N2B and N2A domains, a thick A-band region, and an M-band region where the C-terminus is embedded.
Extensive alternative splicing in the 364 exons of TTN leads to forming various molecular isoforms.Previous studies have shown three main titin isoforms expressed in cardiomyocytes: the adult N2B isoform, the adult N2BA isoform, and the fetal cardiac titin (FCT) isoform.The distinct characteristics of each titin isoform arise from differences in their I-band sequences, while the Z-disk, A-band, and M-line regions are highly conserved across all isoforms 28 .Due to the longer extensible I-band region, the N2BA isoform is more compliant than N2B.The N2BA isoform contains additional spring-like elements in the PEVK and tandem Ig regions, leading to lower passive tension in cardiomyocytes compared to other isoforms [29][30][31] .
Molecular structure of sarcomere and the interaction of Titin with thin and thick filaments is demonstrated in Fig. 1.

Z-disk
The Z-disk region spans 826 amino acids horizontally across the structure and contains seven Ig domains separated by Z-insertion sequences.As the site of numerous structural and functional interactions with myofibrillar and sarcolemmal proteins, the Z-disk is critical for myofibril assembly, stability, and signaling.Z-disks anchor essential proteins like titin-Tcap (telethonin), which enables key Z-disk functions including mechanosensing.Mechanosensing involves recruiting other interacting and signaling partners to the Z-disk in response to mechanical stimuli.Overall, Z-disks play indispensable roles in anchoring titin and enabling vital structural and sensory functions [32][33][34] .www.nature.com/scientificreports/ The Z-disk interacts with small ankyrin proteins, spectrin, desmin, and obscurin, connecting it to other cytoskeletal structures.Filamin C links the Z-disk to costameres via integrins and sarcoglycans, participating in mechanosensory pathways.Additionally, the Z-disk binds nebulin, which helps stabilize Z-disk anchorage through interactions with actin, desmin, CapZ and myopalladin.α-Actinin binding also enhances Z-disk mechanical stability.Overall, the Z-disk forms critical protein interactions that provide structural support and sensory functions [35][36][37][38][39][40] .

I-band
The I-band region of titin displays extensive alternative splicing, generating diverse isoforms that confer tissuespecific mechanical properties in cardiac and skeletal muscles.Through alternative splicing mechanisms, a spectrum of isoforms emerges, tailoring titin's mechanical functions to meet the needs of different muscle types.The I-band thus acts as a central adapter, converting titin into specialized molecular springs via splicing variability.This interactive segment contains a meta-transcript with principal cardiac and skeletal isoforms.Key components include immunoglobulin folds, the cardiac N2B zone, and the skeletal N2A zone containing nonrepetitive sequences and immunoglobulin domains.The proline-glutamate-valine-lysine (PEVK) domain follows, acting as a spring-like element.Together, the I-band components enable the elasticity of titin 38,41 .
The I-band region has distinct proximal and distal segments with specialized roles.The proximal I-band maintains sarcomere integrity, while the medial/distal I-band acts as a bidirectional molecular ruler setting resting length and passive tension 42 .The I-band also functions as a biochemical stress sensor through interactions with αβ-crystallin, a chaperone that stabilizes I-band immunoglobulin domains.Additionally, metabolic enzymes like DRAL, FHL1, and FHL2 associate with I-band sarcomere regions via the Gαq-MAPK pathway 37,43,44 .Indeed, though I-band interactions with the Ca +2 -dependent proteases Calpain-1 and Calpain-3, I-band not only contributes to a sarcomeric quality control pathway but also serves as a reservoir for inactive forms of Calpain-3 45,46 .

A-band
The A-band spans the sarcomere from M-line to M-line, containing thick filaments of myosin.Within the A-band, titin forms a network that maintains the structural integrity of the thick filaments and regulates their length.The A-band exclusively contains fibronectin type III (FnIII) motifs.Immunoglobulin (Ig) and FnIII motifs are arranged in two super-repeats bisected by Ig folds.Unlike the elastic I-band, the A-band is inextensible, providing myosin binding sites that function as stable anchors.A-band super-repeat domains interact with and position sarcomeric myosin binding protein C (MyBP-C).The A-band also contains binding sites for muscle ring finger proteins MURF1 and MURF2.MURF1 likely facilitates quality control and protein turnover at the sarcomere center, while MURF2 interactions aid formation of mature A-band structures 36,38 .

M-band
The M-band integrates structural, signaling, metabolic and protein quality control functions.It contains a putative serine/threonine kinase domain and immunoglobulin cross-hatched rectangle (CII) domains interspersed with M-insertion sequences 47 .While its kinase activity is debated, the M-band kinase domain likely participates in stress sensing through Ca 2+ -calmodulin-regulated mechanochemical signaling 38,48 .During sarcomerogenesis, myomesin constructs an M-band scaffold linking titin to myosin thick filaments, establishing the myomesin-titinmyosin stability axis 49 .The M-band also senses metabolic stress via ligands DRAL/FHL2 that tether metabolic enzymes, and enables ubiquitin-mediated turnover through interactions with nbr1, p62, MURF1 and MURF2 50 .MURF2 binding facilitates M-band's role in cardiac development 51 .Additionally, the extreme C-terminal TTN/ calpain-3/p94 interaction participates in M-band-associated protein turnover 37,52 .

The molecular function of titin
Since the discovery of titin, the complexity and diverse functional roles of titin in health and disease continue to emerge.As the third filament system of the sarcomere alongside actin and myosin, titin forms a unique filament network in cardiomyocytes that engages in mechanical and signaling roles 10 .During muscle development, titin likely controls the assembly of actin and myosin contractile proteins, regulating sarcomere size and thick filament structure.In mature muscle, titin contributes to elasticity mechanisms affecting sarcomere resting lengths and tension-related processes 25 .
The enormity and intricate three-dimensional structure of titin provides structural support to maintain sarcomere integrity during contraction while generating passive tension during stretching.Additionally, the numerous titin-binding proteins arranged in signaling hotspots allow titin to participate in mechanosensing and signal transduction 26,53 .Thus, titin has multifaceted roles beyond viscoelastic force generation: (a) centering thick filaments for optimal active force; (b) assembling sarcomeres; (c) mechanochemical signaling through binding partners; and (d) potentially enabling length-dependent activation underlying the Frank-Starling law 54 .

Comparative analysis of TTN variants
In this study we found 611 distant TTN variant which were not benign and they were pathogenic, likely pathogenic or variant of uncertain significance (VUS).
85% of the variants were reported in exon fragments, while 15% were reported in intron fragments.In ACMG classification, 69.6% of the variants were classified as Pathogenic, 21.6% as Likely Pathogenic, and 8.8% as Variants of Uncertain Significance (VUS).Substitution accounted for 57.25% of the variants, deletion for 29.62%, duplication for 7.36%, and insertion for 5.72%.
The majority of variants occurred in the interval from exon 200 to the end of the molecule, with the hotspot regions identified at exon 326 and 358 being the most common points for variations (Fig. 2).
Most pathogenic variants are located after the exon 326 to the end of the molecule which has higher CADD number compared to others (Fig. 3A).The Genomic Evolutionary Rate Profiling (GERP) score is used to compare the gene nucleotides among the species in the TTN gene 24 .It is supposable that the nucleotides and exons which are conserved in the evolution, can be considered a vital element for survive and loss of function of these components are associated with death and the prevention of its inheritance.In the comparison of the conservity of the gene nucleotides, it can be concluded that most the variants have a notable GERP score which indicates their conservity (Fig. 3B).
In comparing the average CADD score of various exons, it can be concluded that exons with higher CADD scores are located in the end of the gene and the middle part of the gene, the average CADD score is not notable.The first few exons of the gene have a higher CADD score but in the last exons, the CADD score is increased considerably especially in the last 50 exons.VUS variants have less CADD score and likely pathogenic variants also have lesser scores compared to pathogenic variants (Fig. 3C,D).
In the comparing type of genetic alternation in variants, it can be concluded that the most common alternations are substitution and deletions.Most of the deletions have high score numbers while substitutions have various CADD scores.Most of the insertion and duplications also have notable CADD score because of frameshift events while in the substitutions we can observe some lesser CADD score which is not exists in other types of alternations.As demonstrated, most of the pathogenic variants in the first parts of the gene are deletions but the most pathogenic variants in the last parts of the gene have substitutions (Fig. 3E,F).I-band and its isoforms in cardiac compliance and DCM Protein composition patterns can change among different populations and even in various stages of human life.The isoform transforming of sarcomeric proteins in the troponin complex, Myosine heavy chain (MHC), Myosine light chain (MLC) and Titin from fetal to adult through transcriptional changes or alternative splicing is the essential element of myofibril maturation 59 .
A study by Lahmers et al. 59 revealed that fetal titin isoforms are expressed in neonates, containing additional spring elements in the tandem Ig and PEVK regions.This leads to lower stiffness compared to adults, explained by the unique spring composition of fetal cardiac titin in neonates.Changes in titin expression during development likely impact functional transitions and diastolic filling as the heart matures.The fetal cardiac titin isoform, with its extra Ig and PEVK spring elements, gradually disappears postnatally in a species-dependent manner.
In the human heart, the ratio of titin isoform expression is established based on passive tension.There is a high correlation between titin-based passive tension and I-band region size, with lower tension associated with a larger, more elastic I-band.In healthy adult hearts, the N2BA and N2B titin isoforms express at 30-40% and 60-70% respectively.The relative levels of these two isoforms are a key determinant of cardiomyocyte stiffness 60 .Titin plays a central role in the passive ventricular tension.Animal studies have proved that the N2BA isoform is present in the near-term fetus 6 days before birth but after birth disappears and is replaced by a smaller N2B isoform, which predominates in 1-week-old neonate and adults.Adult cardiomyocytes have 15 times more passive tension compared to fetal cardiomyocytes which is confirmed by immunofluorescence microscopy.This transformation is compatible with the heart's function in each stage of life which after birth needs more passive tension to pump the blood effectively through the vessels 61 .
Alternative splicing of the TTN gene plays significant roles in cardiac diseases like dilated cardiomyopathy (DCM).In DCM, the more compliant N2BA isoform is upregulated, decreasing passive stiffness and increasing chamber compliance.Overall, variable expression and splicing of titin isoforms critically influence myocardial passive tension and compliance 30,31,62,63 .
Hidalgo et al. 64 conducted sophisticated experiments to identify the mechanisms influencing myocardial passive stiffness by modifying the phosphorylation state of titin.The study revealed that titin serves as a substrate not only for protein kinase A but also for protein kinase G and protein kinase C α (PKCα).The researchers pinpointed the PEVK region of titin as the primary site for PKCα phosphorylation, demonstrating that phosphorylation at this site enhances passive tension in the myocardium.

Novex variants and tiny titin results alternative splicing
The whole sequence of the human TTN gene contains three isoform-specific mutually exclusive exons named novel exons (novex), which encode for the I-band sequence.Novex1 is presented in exon 45, novex-2 is located in exon 46, and novex-3 is placed in exon 48.The novex-1 and novex-2 Titin isoforms are encoded by transcripts that either include the novex-1 or novex-2 exons.Early stop-gain codon in the novex-3 transcript produces a remarkably tiny isoform (700 kDa) known as novex-3 Titin.The 'tiny Titin' isoform, expressed in all striated muscles, stretches from the Z-disc to the novex-3 domain (C-terminus).Therefore, stress-induced sarcomeric rearrangement may be mediated by novex-3 Titin because of its regulatory involvement in calcium level and GTPase-associated myofibrillar pathways 65 .Furthermore, novexes 2 and 3 may be linked to DCM or ARVC based on the expression levels of novex variations in human cardiac tissues affected by cardiomyopathies.Previous research suggests that novex variations may be attributable to cardiomyopathy 66 .

Splicing regulation of alternative splicing
Encoding Titin by a single gene into various forms is the result of different mRNA splice pathways which leads to Titin isoform classes 57 .The titin gene contains 409 introns, enabling generation of 57 distinct mRNA transcripts through extensive alternative splicing.These include 29 unspliced forms and 28 spliced isoforms.Additional diversity arises from 5 alternative promoters, 9 non-overlapping final exons, and 9 verified polyadenylation sites.The resulting mRNAs vary in: 3' end truncations, 5' end truncations, presence/absence of 173 cassette exons, overlapping exons with different borders, and splicing versus retention of 3 introns 67 .RBM20 regulates a subset of genes involved in developing the heart's muscles by modulating their mRNA alternative splicing.Titin, known to undergo extremely complex alternative splicing, is one of the RBM20's targets.RBM20 specifically manipulates alternative splicing within the I-band of TTN pre-mRNA, which possesses the highest frequency of the alternative splicing process.It has been demonstrated that some alterations in the protein can produce pathogenic TTN isoforms, which are believed to lead to DCM 68 .Surprisingly, Khan et al. 69 , detected 80 distinct circRNAs among nearly a thousand from human hearts, indicating that the I-band of Titin is a hotspot region of circRNAs.Remarkably, the introns on each side of the back-spliced junctions were enriched in RBM20 binding sites, and the introns related to the TTN circRNAs had a five-fold higher frequency of RBM20 binding sites compared to a control set of introns.Studies on the RBP20 knock-out animals, and a cardiac sample of heterozygous RBM20 mutation carrier with substantially compromised synthesis of TTN circRNAs, both provided evidence that RBP20 is involved in the biogenesis of these TTN circRNAs 69 .Furthermore, the

The role of TTN variants in cardiomyopathies
Heterozygous mutations in TTN are commonly associated with cardiomyopathies and TTN has been reported as the most common gene involved in cardiomyopathies 71 .The mutations can be broadly classified into two categories, which are truncating or missense mutations.Truncating mutations lead to premature termination of Titin protein synthesis, resulting in either an altered protein or the loss of functional domains.In contrast, missense mutations result in the replacement of amino acids, potentially causing interference with the typical operation of the Titin protein 36 .
The ongoing inquiry into the exact molecular mechanisms by which TTN mutations lead to cardiomyopathies illuminates the intricate relationship between TTN mutations and various forms of cardiomyopathies.The haploinsufficiency model is a notable mechanism that proposes the presence of truncating mutations in one allele of the TTN gene results in a reduction in Titin expression, consequently inducing a functional deficit of Titin protein.The phenomenon mentioned above possesses the capability to disrupt the sarcomere assembly process, alter the mechanical properties of cardiac muscle cells, and prevent the heart's contractile function, leading to the manifestation of cardiomyopathy.Another proposed mechanism which even can be manifest in dominant pattern is missense mutations.This occurrence takes place when the mutated form of the Titin protein impairs the normal functioning of the unaltered Titin protein, leading to compromised assembly and operation of the sarcomere.
Moreover, it is plausible that TTN mutations may trigger aberrant splicing occurrences, leading to the production of deficient or abnormal Titin isoforms, thus playing a role in the pathogenesis of cardiomyopathy c.The bioinformatics analysis of reported variants in TTN related to cardiomyopathies has been shown in Table 1.

Dilated cardiomyopathy
Idiopathic factors are just as significant in the pathophysiology of DCM as acquired variables (such as infections, poisons, or autoimmune diseases).Individuals harboring TTN mutations exhibit a higher susceptibility to developing DCM compared to other forms of the disease 36,[72][73][74] .Idiopathic DCM, including familial and sporadic instances, has a genetic etiology, according to a vast number of studies 75,76 .
A review study by Chauveau et al. 26 reported that Among the TTN mutations linked to DCM, 29 are categorized as nonsense mutations, with three of them occurring in the I-band, while the remaining 26 are located in the A-band.Additionally, 17 frameshift mutations are reported, with three in the I-band and 14 in the A-band.Furthermore, 18 mutations are predicted to affect TTN splicing TTN mutations, particularly truncating variants (TTNtv) in the A-band region and in exons that are highly utilized across the range of titin isoforms, have been shown in a number of studies to be strongly associated with the occurrence of DCM and its severity, accounting for the majority of cases [77][78][79][80] .
Although fewer TTNtv have been identified in pediatrics, a study by Fatkin et al. 81 on the young population showed that the prevalence between adolescents and adults is similar, indicating that they need to have multiple clinical and genetic risk factors other than a single TTNtv to present with CDM.TTNtv accounts for 25% of familial cases and 18% of sporadic cases of idiopathic dilated cardiomyopathy 82 .The aforementioned TTNtv have demonstrated a remarkably low prevalence within the broader populace.
According to Fatkin et al. the prevalence of TTNtv is 20% among individuals with DCM, whereas only 0.5% of the general population carries this type of mutation 83,84 .The aforementioned data aligns with the results of Fang et al. 85 survey, which indicated an overall prevalence rate of 17%.The survey also revealed that 23% of cases were familial, while 16% were sporadic.For example, mutations in the A-band are implicated as predominant genetic causes of DCM [86][87][88] .
An important question is how minor TTNtv carrier populations can avoid presenting with DCM.A convincing explanation comes from a study by Roberts et al. 77 showing that the two major adult cardiac titin isoforms, N2BA and N2B, are responsible.These abundant full-length isoforms predominantly contain distal A-band exons, where most DCM-causing TTNtvs are located.However, mutations in proximal exons not present in all TTN transcripts do not cause DCM.

Hypertrophic cardiomyopathy
HCM is the most common inherited cardiomyopathy, frequently arising from sarcomere gene defects.Characterized by arrhythmias and heart failure symptoms due to left ventricular outflow obstruction, diastolic dysfunction, ischemia, or mitral regurgitation, HCM displays autosomal dominant inheritance.Mutations, predominantly missense, in one or more sarcomere genes underlie most cases of HCM.To date, over 1400 mutations have been identified in genes encoding primarily sarcomeric proteins 89 .
Due to the involvement of a vast range of mutations with distinctive penetrance, a comprehensive understanding of the pathophysiological mechanisms underlying the development of HCM in the presence of sarcomererelated gene mutations is still unfulfilling 90 .In a study conducted by Ingles et al. 91 on 33 genes reported to have an association with HCM, only 8 genes (MYBPC3, MYH7, TNNT2, TNNI3, TPM1, ACTC1, MYL2, and MYL3) were shown to have a definitive impact on occurring HCM.It is estimated that around 30% of HCM patients have unidentified genes responsible for the condition.www.nature.com/scientificreports/ The gene MYBPC3, which codes for cardiac myosin-binding protein C, is the most important gene in this process accounting for up to half of the mutations identified [92][93][94] .In the second place, MYH7, which is responsible for encoding the beta-myosin heavy chain, is present in approximately 15-25% of patients diagnosed with HCM 92,95 .
In comparison to other plausible etiologies of HCM, the presence of the TTN gene mutations exhibits a relatively low ranking.Several studies reported four TTN variants resulting in gain-of-function effects in HCM patients.Satoh et al. 96 found a Z-line mutation (c.2219G > T, p.Arg740Leu) which increases alpha-actinin binding affinity.Two studies, similarly, reported a mutation in cardiac-specific N2B exon 49 [c.12347C > A, p.Ser4116Tyr] resulting in increased TTN binding to DRAL/FHL2 97,98 .The TTN/T-CARP interaction is reinforced by the presence of two mutations located in exons 103 and 104-N2A, c.29231G > A, p.Arg9744 (initially reported as p.Arg8500His) and c.29543G > A, p.Arg9848Gln (initially reported as p.Arg8604Gln), as reported by Arimura et al. 99 .Lopes et al., in a different study, reported 219 TTN variants in a population of unrelated HCM patients.Of those 87% coexisted with mutations in HCM-related sarcomere gene defects and only 13% were found isolated 26,100 .However, in a study on 90 HCM patients and their close relatives, the mutation screening revealed no clue of the TTN gene being involved in their pathogenesis 101 .Similarly, Martijn Bos et al. 102 detected no TTN mutation in a group of 389 HCM patients.

Restrictive cardiomyopathy
Restrictive cardiomyopathy is a diverse collection of disorders that primarily affect the myocardium, with a lesser impact on the endocardium and sub-endocardium.It is characterized by increased stiffness of the ventricular walls leading to restricted ventricular filling, which consequently results in significant diastolic dysfunction, elevated end-diastolic pressure, and reduced ejection fraction in the advanced stages 103,104 .
The epidemiology of this disease is not well understood in the literature due to classification and etiology reporting difficulties, but RCM is surely the least common form of cardiomyopathies, representing 2% to 5% of cases 2,105 .There are a variety of diseases that can cause it, including infiltrative disorders like amyloidosis and sarcoidosis, non-infiltrative disorders like diabetes and scleroderma, storage disease, endomyocardial disease, and cardiotoxicity brought on by chemotherapy or radiotherapy 2 .
The role of TTN variants in RCM is relatively unknown and more investigations are needed to illustrate this fact.In 2013, for the first time, Peled et al. discovered a novel missense mutation (c.50057A > G, p.Tyr16686Cys) in the intersection of the A and I regions of Titin (IA junction).This mutation was found to play a role in earlyonset familial RCM, which affected six members of a family.They asserted that Titin determines the sarcomere's resting tension, and their study offers genetic proof of its critical significance in diastolic function. 36,108,109.In another study, Kizawa et al. 110 found another novel TTN missense mutation (c.22769C > A, p.P7590Q) in a young boy with neurofibromatosis type 1, which is thought to be responsible for RCM co-occurrence.This de novo mutation is also located at the IA junction.

Arrhythmogenic right ventricular cardiomyopathy (ARVC)
Arrhythmogenic cardiomyopathy (ACM), is a rare and potentially life-threatening heart muscle disease with a prevalence of approximately 1:1000 to 1:5000 [111][112][113] .Although asymptomatic in most instances upon diagnosis, it is characterized by palpitations, atypical chest pain, and syncope caused by cardiac arrhythmia, mostly in the right ventricle, which leads to the term "arrhythmogenic right ventricular cardiomyopathy (ARVC)" [114][115][116] .This condition is characterized by the progressive replacement of the myocardium with fibrofatty tissue, a process that begins at the epicardium, turns into a regional wall motion abnormality, and eventually spreads throughout the myocardium, resulting in the development of ventricular dilation and multiple aneurysms [117][118][119] .
The primary etiology of ACM is attributed to mutations in genes that encode desmosomal proteins, mainly with an autosomal dominant pattern of inheritance and over 30 percent of cases being familial.JUP, DSP, PKP2, DSG2, and DSC2 genes are the most probable to be involved.LMNA and TMEM43 are two additional genes that have been linked to the nuclear envelope, and there are genes that are shared with other cardiomyopathies (such as DES, PLN, TGFB3, TTN, and SCN5A) 112,[120][121][122][123] .

Frequent TTN-related molecules in cardiomyopathies
There are several molecules which play a considerable role in the signaling and function of Titin.In the present study, we evaluated their interaction with Titin and consider their interaction with Titin in the pathogenesis of cardiomyopathies (Fig. 4).

Calpain
Calpain, a family of Ca 2+ -dependent cytosolic cysteine proteases, plays a role in various cellular processes, including cell death and tissue remodeling 126 .It has been implicated in several cardiac conditions, including dilated cardiomyopathy, alcohol-related cardiomyopathy, chemotherapy-induced cardiomyopathy, arrhythmogenic cardiomyopathy, and diabetic cardiomyopathy [127][128][129][130][131] .Sustained over-expression of calpain-2, specifically in cardiomyocytes, induced age-dependent dilated cardiomyopathy in mice 127 .www.nature.com/scientificreports/MuRF1/2 Muscle ring finger (MuRF) proteins are muscle-specific ubiquitin E3 ligases that regulate the ubiquitin-proteasome system and modulate cardiac mass and function 132 .A study by Su et al. 133 showed a higher prevalence of rare MuRF1 and MuRF2 variants in hypertrophic cardiomyopathy (HCM) patients compared to controls.HCM patients with these rare MuRF1/2 variants were younger and had greater maximum left ventricular wall thickness than those without the variants 133 .
ERK ERK (Extracellular signal-regulated kinase) plays a central role in cardiac physiology and hypertrophy [134][135][136] .ERK signaling is implicated in various forms of cardiac hypertrophy and progression to heart failure 135 .Altered ERK activity has been linked to HCM 134 .ERKs are considered key regulators of cardiac hypertrophy since they are activated by most, if not all, stress stimuli known to induce hypertrophic growth 137 .Studies show that concurrently eliminating ERK1 and ERK2 in the heart leads to eccentric hypertrophy with chamber dilatation and cardiomyocyte elongation 136 .

NFAT
Nuclear factor of activated T-cells (NFAT) transcription factors are implicated in developing cardiac hypertrophy and heart failure 138 .Activation of NFAT signaling induces pathological remodeling of cardiomyocytes 139 .Inhibition of NFAT prevents maladaptive cardiac growth in response to stress stimuli 140 .Targeting NFAT signaling pathways may be therapeutic for specific cardiomyopathies 141,142 .

FHL1/2
Mutations in the four-and-a-half LIM domain proteins 1 and 2 (FHL1 and FHL2) are associated with reducing body myopathy and hypertrophic cardiomyopathy 143 .FHL1/2 are involved in sarcomere assembly and signaling and highly expressed in skeletal and cardiac muscle 144,145 .Abnormal FHL proteins cause structural defects in sarcomeres and impaired muscle contraction 146 .FHL1 mutations account for 8-10% of familial reducing body myopathy cases which can include cardiomyopathy 147,148 .Chu et al. 145 reported FHL1 upregulation in Cardiac ventricles of two mouse models with cardiac hypertrophy and dilated cardiomyopathy.

MARP
Muscle ankyrin repeat proteins (MARPs), including CARP, Ankrd1/2, and DARP, are a family of ankyrin repeat proteins expressed in striated muscle that are induced by stress.MARPs play regulatory roles in the muscle stress response and hypertrophy pathogenesis 149 .Overexpression of CARP is linked to dilated cardiomyopathy in animal models 150 .In addition, Patients with hypertrophic, dilated, ischemic, and arrhythmogenic right ventricular cardiomyopathy are more likely to develop CARP upregulation 62,149,151,152 .Missense mutations in the Ankrd1 gene have recently been identified as the cause of dilated and hypertrophic cardiomyopathy in humans 99,149,153,154 .CARP modulation of gene expression may contribute to adverse ventricular remodeling in cardiomyopathies 155 .

SRF
Serum response factor (SRF) is a transcription factor regulating cardiac gene expression important for adaptation to stress 160 .SRF inactivation in animal models causes dilated cardiomyopathy 160 .SRF likely controls genes involved in maintaining normal cardiac structure and function 161 .Alterations in SRF-dependent gene regulation may underlie some cardiomyopathies 162 .

MLP
Muscle LIM protein (MLP) is involved in mechanosensing and stretch response in cardiomyocytes 163 .MLP knockout mice develop dilated cardiomyopathy 164 .Loss of MLP leads to impaired myocyte stretch signaling and contraction 165 .MLP deficiency is implicated in some forms of familial dilated cardiomyopathy 166 .

MyBP-C
Myosin binding protein C (MyBP-C) is important for maintaining sarcomere structure and regulating muscle contraction 167 .Mutations in cardiac MyBP-C are the most common cause of hypertrophic cardiomyopathy 168 .Abnormal MyBP-C disrupts sarcomere function leading to reduced contractility and development of hypertrophy 169 .

Myomesin
Myomesin is a major component of the sarcomeric M-band involved in thick filament organization 170 .Myomesin mutations have been associated with hypertrophic and dilated cardiomyopathy in some patients 171 173 .Mutations affecting SH2 domains of ZASP/Cypher proteins are linked to dilated cardiomyopathy 174 .Disruption of ZASP protein interactions likely impairs structural organization and signaling processes in cardiac muscle 175 .

Ras
Ras family small GTPases regulate growth and survival signaling 176 .Constitutively active mutant Ras expressed in mouse hearts causes dilated cardiomyopathy phenotype 177 .Hyperactive Ras leads to increased cell growth, altered metabolism and myocardial dysfunction 178 .

Raf
Raf kinases act downstream of Ras to activate MEK/ERK signaling involved in cell proliferation and differentiation 179 .Cardiac-specific expression of activated Raf in transgenic mice induces dilated cardiomyopathy 180 .

Alpha actinin
Alpha-actinin-2 (ACTN2) is the sole muscle isoform of α-actinin expressed in cardiac muscle 181 .Previous studies have shown that novel ACTN2 variants are associated with familial HCM 182 .Previous studies have shown that novel ACTN2 variants are associated with 181 .Mutations in ACTN2 have been linked to mild to moderate forms of HCM 181 .Disease modeling of an ACTN2 mutation has guided clinical therapy in HCM 183 .Genome-wide analyses have also demonstrated that ACTN2 mutations can cause HCM 184 .

Filamin C
In striated muscle, different forms of the Ank3 gene product (ankyrins-G) are produced due to tissue-specific alternative splicing.These ankyrins-G have a shared segment called the Obscurin/Titin-Binding-related Domain (OTBD), which is consistent across ankyrin genes and links obscurin and Titin to Ank1 gene products.Previously, it was suggested that the OTBD segment in ankyrins plays a unique role in muscle protein interactions.In recent studies, muscle proteins that can bind to the ankyrin-G OTBD were identified as plectin and filamin C, both crucial for muscle development and structure.These three proteins (ankyrin-G, plectin, and filamin C) are found together in skeletal muscle and are observed in the same regions (costameres) of adult muscle fibers 185 .Filamin C (FLNC) is an actin-binding cytoskeletal protein encoded by the FLNC gene, instrumental in maintaining sarcomeric integrity.While first identified as causative in myofibrillar myopathy, recent evidence reveals a key role for FLNC in cardiomyopathy pathogenesis.Truncated FLNC variants predominate in DCM and ARVC, while non-truncated forms are more common in hypertrophic cardiomyopathy and restrictive cardiomyopathy.The primary mechanisms underlying FLNC-associated cardiomyopathies are protein aggregation from nontruncating mutations and haploinsufficiency resulting from filamin C truncation 186 .

Nebulin
Members of the nebulin protein family, which includes nebulin, nebulette, LASP-1, LASP-2, and N-RAP, are diverse in size, expression pattern, and function, but they all bind to actin.While nebulin's presence in the heart is minimal, nebulette stands out for its heart-specific expression.Crucially, mutations in the nebulette gene have been linked to DCM.Transgenic mice with these mutations display symptoms that mirror this human heart condition 187 .

Mechanosensory signaling mechanism of titin
Titin plays a crucial role in mechanosensing, which is the ability of cells to sense mechanical forces.When muscles undergo stretch or contraction, Titin is subjected to mechanical stress and strain.This mechanical deformation of Titin can trigger mechanotransduction pathways, converting mechanical signals into biochemical signals.These pathways involve the activation of various signaling molecules, including kinases, phosphatases, and transcription factors, leading to cellular responses such as gene expression changes, protein synthesis, and remodeling of the contractile apparatus 188 (Fig. 4).

Z disk region
The Z-disc region of Titin consists of Z-repeats and Ig-domains Z1 and Z2, forming the very NH2-terminal end.Telethonin connects two Titin molecules from one sarcomere, which is essential for sarcomere integrity.Cardiac telethonin undergoes phosphorylation by various kinases and mutations in telethonin are linked to various cardiac cardiomyopthies.Some mutations might disrupt its phosphorylation and, thus, its function.Telethonin interacts with the muscle LIM protein (MLP), together with actinin, MLP, Titin, and telethonin might form a complex that senses mechanical stretch 50 .

N2-B region
Cardiac-specific N2-B region which made up of Ig-domains can bind to two isoforms of the LIM domain protein, FHL-1 and FHL-2 which respond strongly to biomechanical stress, and can move to the nucleus to work as transcriptional co-activators.FHL-2's activity could suppress calcineurin, inhibiting pathological cardiac growth while FHL1 might connect to the MAPK signaling cascade.Under non-stimulating conditions, MEK1/2 anchors ERK in the cytoplasm, but after activation, it shifts ERK to the nucleus, activating specific transcription factors.
ERK2 has been seen to phosphorylate Titin's N2-Bus sequence, potentially affecting myofilament stiffness.Knocking down FHL1 in mice changed myofibrillar responsiveness and reduced hypertrophic signaling.Hence, the N2-B/FHL-1/MAPK complex might be a key biomechanical stress sensor in cardiomyocytes 44,58,137,189,190 .

M-band region
The M-band region of Titin, particularly the Titin kinase (TK) domain, is a significant area for hypertrophic signaling.TK's conformational changes, suggesting its role as a biomechanical stress sensor, might be biomechanically induced.When activated, TK interacts with Nbr1, forming a complex with p62/SQSTM1 and muscle-specific ubiquitin E3 ligases MuRF1, MuRF2, and MuRF3.
The TK signaling complex with the zinc-finger protein nbr1 is involved in mechanically-activated signaling.Nbr1 directs the ubiquitin-binding protein p62/SQSTM1 to sarcomeres where it interacts with the musclespecific E3 ligase MuRF2, linked to the transactivation domain of serum response factor (SRF).Mechanical inactivity triggers MuRF2 nuclear migration, decreasing nuclear SRF and suppressing transcription.Mutations in the TK domain disrupt this mechanism, resulting in hereditary muscle disorders 50,191 .
Of course, it should be considered that subsequent investigations have proposed that TK functions as an inactive pseudokinase, utilizing its kinase scaffold to recruit MuRF1 for biomechanically regulated autophagy pathways 192,193 .

The hotspot region for TTN variants
In a quantitative analysis of variants, it was revealed that the most common hotspot region for variants is the exon number 326 which is located in the A band as the Fibronectin type III domain 194 and has a more considerable number of variants compared to other parts which are followed by exon 358 (containing Ig-like domain and Fibronectin type III domain) 194 and exon 48.Among the introns, intron 47 can be considered as the hotspot point for variants compared to other introns 194 (Fig. 2).

Discussion
This study identified 611 distant TTN variants, classified as pathogenic, likely pathogenic, or variants of uncertain significance (VUS).These variants predominantly occurred in exon fragments (85%), with 69.6% classified as pathogenic, 21.6% as likely pathogenic, and 8.8% as VUS in ACMG classification.Substitutions accounted for 57.25% of the variants, deletions for 29.62%, duplications for 7.36%, and insertions for 5.72%.The majority of pathogenic variants were located after exon 326, exhibiting higher CADD scores.GERP scores indicated conservity among gene nucleotides, with most variants having notable GERP scores.Exons at the end of the gene displayed higher average CADD scores.VUS variants had lower CADD scores.
TTN, a functionally and structurally essential component of striated muscles, is the largest human protein 10,11 .It consists of four functional regions including N-terminal, I-band, A-band, and C-terminal 26 .The N-terminal is an anchor for Z-disk, which not only plays a crucial role in myofibril assembly and stability but also in sensory functions, protein interactions, and signaling pathways [32][33][34][35][36][37][38][39][40] .Owing to alternative splicing, I-band is the central adopter specializing titin for specific tissues.The elasticity of the titin is mostly attributable to the I-band unit 38,41 .On the contrary to the I-band, the A-band is not extensible and is a stable anchor for myosin fibers.It also interacts with various proteins contributing to protein turnover at the sarcomeric center 38,41 .The M-band constitutes the myomesin-titin-myosin and also senses and responds to the metabolic stress 50 .
The passive tension of the human heart is determined by the pattern of expression of titin isoforms.Expression of more elastic and larger I-band isoforms is associated with lower titin passive tension.The ratio of N2BA and N2B isoform expression determines the stiffness of cardiomyocytes 60 .If the balance between N2BA and N2B is disrupted and N2BA isoform upregulates, the decrease in passive stiffness of the heart brings about DCM 30,31,62,63 .Mutations in the TTN gene are speculated to bring about cardiomyopathies through disruption in sarcomere assembly or contractility, or triggering aberrant splicing 30,31,62,63 .
In accordance with our study, another study demonstrated that most TTN variants associated with TTN are located in the A-band unit followed by the I-band 26 .Truncating TTN variants located in the A-band region are the predominant TTN mutations associated with the DCM [77][78][79][80][86][87][88] . The N2A and N2B isoforms contain distal exons of the A-band.Therefore, variants affecting the A-band and its distal regions are more frequently reported to manifest with DCM, while, the N-terminal mutations are less likely to bring about DCM, considering they are not expressed in N2BA and N2B isoforms 77 .
TTN mutations are not as prominent in HCM compared to DCM.HCM is speculated to arise from mutations in sarcomere-related genes; nonetheless, the exact pathophysiology of HCM is yet to be found 90 .Mutations in Sarcomeric, non-sarcomeric, and sarcomere-associated proteins are proposed to contribute to the development and inheritance of RCM 1,2,106,107 .Although the role of TTN variants in the pathogenesis and inheritance of RCM is not fully understood, it is known that titin is the key determinant of sarcomere resting tension and diastolic function 36,108,109 .Similarly, the impact of TTN mutations in ARVC is not yet determined.However, rare TTN variants have been reported in probands and family members of ARVC patients 121,123 .
The most common hotspot for mutations is exon 326 of the TTN gene which is located in the A-band region.Notably, the exon containing the most TTN variants is 358, also in the A-band.As presented, the TTN variants were primarily located in a small number of exons which are mostly situated at A-and I-bands.This localization of TTN variants might stem from the higher fatality of mutations in other locations, or conversely, these mutations do not exhibit clinical symptoms to prompt genetic evaluation.
The conservatory TTN exons seem to be associated with the pathogenicity of the variants This might be explained, at least in part, by the theory that more conserved nucleotides could be essential, and mutations affecting this nucleotide could be more pathogenic.

Figure 1 .
Figure 1.Molecular structure of sarcomere and the interaction of Titin with thin and thick filaments.

Figure 2 .
Figure 2. Prevalence of variants in different exons and introns in TTN.

Figure 3 .
Figure 3. Comparative analysis of TTN variants with their pathogenicity, type of alternation, and conservity.

Figure 4 .
Figure 4. Illustration of the intricate signaling pathway implicated in the development of cardiomyopathy associated with Titin and other related proteins.

Table 1 .
Bioinformatics analysis of Pathogenic, Likely pathogenic, Unknown Significance reported variants in TTN related to cardiomyopathies.
125e to the conclusion that this factor was altered in cardiomyopathies such as DCM and ARVC.Beyond cardiomyopathies, TTN mutations are implicated in numerous non-cardiac muscle disorders.According to Chauveau et al.26, 39 TTN mutations have been identified so far in four pure skeletal muscle myopathies: limb girdle muscular dystrophy type 2J (LGMD2J), late-onset autosomal dominant tibial muscular dystrophy (TMD), hereditary myopathy with early respiratory failure (HMERF), and congenital centronuclear myopathy (CNM).Additional conditions associated with TTN variants include early adult onset recessive distal titinopathy, earlyonset myopathy with fatal cardiomyopathy, multi-minicore disease with heart disease, childhood-juvenile Emery-Dreifuss-like phenotype without cardiomyopathy, and adult-onset recessive proximal muscular dystrophy125.